Structural basis of TRPC4 regulation by calmodulin and pharmacological agents
- Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Germany
- Department of Neurophysiology, Physiological Institute, Julius-Maximilians-Universität Würzburg, Germany
- Goldfinch Bio, United States
Figures
Figure 1
The effect of three pyridazinone-based small molecules on TRPC4 and on (-)-Englerin A (EA)-induced opening of TRPC4.
(A, E and I) TRPC4-expressing Xenopus oocytes were held at −40 mV and perfused with increasing concentrations of GFB-9289 (A), GFB-8438 (E) or GFB-8749 (I) to test the potential activation effect. …
Figure 2 with 5 supplements
Cryo-EM structure of inhibitor-bound TRPC4 channel.
(A) Side and top view of the cryo-EM map of GFB-8438 inhibitor-bound TRPC4, with each subunit colored differently. Positions of the transmembrane domain (TMD) and intracellular cytosolic domain …
Figure 2—figure supplement 1
Cryo-EM image processing workflow for TRPC4 in complex with the inhibitor.
(A) The top panel shows a representative digital micrograph area and selected 2-D class averages of inhibitor GFB-8438 bound TRPC4. Scale bars, 50 nm and 10 nm, respectively. The initial refinement …
Figure 2—figure supplement 2
Cryo-EM map of TRPC4 in complex with the inhibitor GFB-9289 and GFB-8749.
(A) Side and top view of the cryo-EM map of GFB-9289 inhibitor-bound TRPC4, with each subunit colored differently. Positions of the transmembrane domain (TMD) and intracellular cytosolic domain …
Figure 2—figure supplement 3
Local resolution of the TRPC4-ligand complex maps.
Maps of GFB-8438, GFB-9289 and GFB-8749-bound TRPC4, respectively, colored according to the local resolution. Representative regions of the density with the fitted atomic model are shown below the …
Figure 2—figure supplement 4
Comparison between different TRPC4 structures.
(A) Structural alignment of a protomer of the inhibitor GFB-8438 bound TRPC4 with that of TRPC4 in its apo state. The protomer of the TRPC4 apo structure is shown in cartoon representation and …
Figure 2—figure supplement 5
Domain architecture of zebrafish TRPC4 channel.
Cartoon representation of a TRPC4 protomer. Each domain is shown in a different color and labeled accordingly.
Figure 3 with 1 supplement
Comparison of the ligand-binding pocket in TRPC4.
(A) Close-up of ligand-binding pocket in the apo TRPC4 structure, which is enclosed by the four helices S1 to S4 of the voltage sensing-like domain. (B) Superposition of inhibitor-bound (red) and …
Figure 3—figure supplement 1
Sequence alignment of zebrafish TRPC4, human TRPC4, TRPC5, and TRPC6.
The highlighted and marked residues denote the conserved residues in TRPC4 and TRPC5 interacting with the inhibitor GFB-8438. The residues highlighted in pink color shows the critical difference …
Figure 4 with 2 supplements
Comparison of the ion conduction pore and Ca2+-binding site.
(A) Side view of the pore-forming region of TRPC4 in the apo- (blue), GFB-8438 (red) GFB-9289 (green) and GFB-8749 (cyan-blue) inhibitor-bound structures. Only the two opposing subunits of the …
Figure 4—figure supplement 1
Different views of the lipid binding pocket at the interface between two subunits.
Phosphatidic acid (pink) that binds at the interface between two subunits near the pore region is shown in stick representation along with the corresponding density. The interacting residues from …
Figure 4—figure supplement 2
Ca2+-binding site in the VSL domain of apo and ligand-bound TRPC4.
(A) Close-up view of the Ca2+-binding site in apo and ligand-bound TRPC4 determined in amphipols. The coordinating residues in the Ca2+ ion binding site and the modelled Ca2+ ion are shown in stick …
Figure 5 with 6 supplements
Structural basis for inhibition of TRPC4 by calmodulin.
(A) One to four CaM molecules are bound to the CIRB binding sites of the tetrameric TRPC4 channel. 13% of particles are decorated with one (yellow), 35% with two (lilac), 31% with three (grey) and …
Figure 5—figure supplement 1
Analysis of CaM binding to TRPC4 by biochemical methods.
(A) SDS gel electrophoresis analysis of the TRPC4 pull down experiment performed with a CaM Sepharose column. Lane 1 - protein size marker, lane 2 - TRPC4 input, lane 3 - flow through, lane 4 - …
Figure 5—figure supplement 2
Cryo-EM image processing of the TRPC4- CaM complex.
(A) The top left panels show a representative digital micrograph area and selected class averages of the TRPC4-CaM complex. Scale bars, 50 nm and 10 nm, respectively. The initial refinement …
Figure 5—figure supplement 3
Local resolution maps of TRPC4-apo (LMNG) and TRPC4-CaM.
(A, B) Maps of TRPC4-CaM and TRPC4-apo (LMNG), respectively, colored according to the local resolution. Representative regions of the density with the fitted atomic model are shown below the local …
Figure 5—figure supplement 4
Cryo-EM image processing and structure determination of TRPC4 solubilized in LMNG.
(A) The top left panels show a representative micrograph and class averages of TRPC4 solubilized in LMNG. Scale bars, 50 nm and 10 nm, respectively. The density resulting from the initial 3D …
Figure 5—figure supplement 5
Comparison of the ion conduction pore and Ca2+-binding site.
(A) Side view of the pore-forming region of TRPC4 in the apo (LMNG)- (blue), and CaM-bound (purple) structures. Only the two opposing subunits of the tetrameric channel are shown as ribbon …
Figure 5—figure supplement 6
Biochemical and structural analysis of CaM N- and C-lobe binding to TRPC4.
(A) SDS gel electrophoresis analysis of TRPC4 binding to MBP-fused N- and C-lobes of CaM after a pull-down assay using amylose resin. Lane M - protein size marker, lane 1 - TRPC4 input, lane 2 - …
Figure 6
Comparison of CaM binding in TRPC and TRPV channels.
(A) Calmodulin (CaM) interacts with the rib helix of TRPC4. Side (upper panel) and bottom (lower panel) view of the CaM-bound TRPC4 is shown, with TRPC4 structure in cartoon representation with …
Figure 7
Model for TRPC4 modulation.
(A) Canonical TRP channels can transiently open to allow the passage of Ca2+ ions into the interior compartment (left panel). Several mechanisms modulate the activity of the channel: binding of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (HEK293 GnTI-) | HEK293 GnTI- | ATCC | RRID:CVCL_A785 | CRL-3022 |
| Cell line (HEK293T) | HEK293T | ATCC | RRID:CVCL_LF41 | |
| Cell line (Sf9) | Sf9 | Oxford Expression Technologies Ltd (UK) | RRID:CVCL_0549 | Cat.No.600100 |
| Gene (Danio rerio) | TRPC4DR | GenScript NCBI Reference sequence: NM_001289881 | ||
| Recombinant DNA reagent | pCDNA3.1+TRPC4ZF | Vinayagam et al., 2018 PMID:29717981 | ||
| Recombinant DNA reagent | pEG BacMam | Eric Gouaux Lab PMID:25299155 | ||
| Recombinant DNA reagent | pEG BacMam +TRPC4 ZF (See methods section for details) | Vinayagam et al., 2018 PMID:29717981 | ||
| Recombinant DNA reagent | pGEMHE 22 | Promega | P2151 | pGEMHE 22 is a derivative of pGEM3z |
| Chemical compound, drug | (-)-Englerin A | Carl Roth | Cat.No.6492.1 | |
| Software, algorithm | SPHIRE software package | Moriya et al., 2017 PMID:28570515 | ||
| Software, algorithm | crYOLO | Wagner et al., 2019 PMID:31240256 | ||
| Software, algorithm | Origin 2020 pro | OriginLab Corporation | ||
| Software, algorithm | TranSPHIRE | Stabrin et al., 2020 doi:https://doi.org/10.1101/2020.06.16.155275 | ||
| Software, algorithm | Chimera | Pettersen et al., 2004 PMID:15264254 |
Table 1
Plunging and imaging conditions used for cryo-EM analysis of TRPC4 bound with ligands.
| 1.1 Plunging conditions | ||||||
|---|---|---|---|---|---|---|
| Sample | Grid type | Volume | Concentration | Blotting time | Blotting force | |
| TRPC4-8438 | C-Flat 2/1 | 3 µl | 0.3 mg/ml | 3 s | −10 | |
| TRPC4-9289 | C-Flat 1.2/1.3 | 3 µl | 0.35 mg/ml | 3 s | 0 | |
| TRPC4-8749 | C-Flat 1.2/1.3 | 3 ul | 0.35 mg/ml | 3s | 0 | |
| TRPC4-cam | QF 2/1 | 3 µl | 0.3 mg/ml | 3 s | 0 | |
| TRPC4-apo(LMNG) | C-Flat 1.2/1.3 | 3 µl | 0.4 mg/ml | 3 s | −3 | |
| 1.2 Imaging Conditions | ||||||
| Microscopy | TRPC4-apo | TRPC4-CaM | GFB-9289 | GFB-8438 | GFB-8749 | |
| Microscope | Titan Krios (X-FEG, Cs-corrected) | Titan Krios (X-FEG, Cs 2.7 mm) | ||||
| Voltage [kV] | 300 | 300 | ||||
| Defocus range [µm] | 0.65 to 3.02 | 0.38 to 3.48 | 0.68 to 3.64 | 0.35 to 3.52 | 0.86 to 3.82 | |
| Camera | K2 counting | K2 counting | K3 Super res. | K3 Super res. | K3 Super res. | |
| Pixel size [Å] | 0.85 | 0.85 | 0.455 /0.91a | 0.455/0.91a. | 0.455 /0.91a | |
| Total electron dose [e/Å2] | 88.7 | 88.2 | 65.45 | 66.58 | 72 | |
| Exposure time [s] | 10 | 10 | 3 | 3 | 3 | |
| Frames per movie | 50 | 80 | 60 | 60 | 60 | |
| Number of images | 2755 | 6937 | 2369 | 4444 | 1260 | |
| (3079) | (7972) | (2970) | (4676) | (1290) | ||
Table 2
Refinement and model validation statistics.
| Refinement statistics | |||||
|---|---|---|---|---|---|
| TRPC4-apo | TRPC4-CaM | GFB- 9289 | GFB-8438 | GFB-8749 | |
| Number of particles | |||||
| used in refinement | 126873 | 160829 | 65811 | 42524 | 44989 |
| Final resolution [Å] | 2.8 | 3.6 | 3.2 | 3.6 | 3.8 |
| Map sharpening factor [Å2] | -57.97 | -72.37 | -100 | -61.35 | -120 |
| Electron dose particles final refinement [e-/Å2] | Polished particles | Polished particles | Polished particles | Polished particles | 72 |
| Model geometry and validation statistics | |||||
| Atomic model composition | |||||
| Non-hydrogen atoms | 22,124 | 21,650 | 21,152 | 21,080 | 21,056 |
| Refinement (Phenix) | |||||
| RMSD bond | 0.008 | 0.011 | 0.008 | 0.007 | 0.011 |
| RMSD angle | 0.738 | 0.983 | 0.645 | 0.771 | 0.736 |
| Model-to-map fit, CC mask | 0.84 | 0.86 | 0.85 | 0.86 | 0.83 |
| Validation Ramachandran plot (%) | |||||
| Outliers | 0.0 | 0.04 | 0.0 | 0.0 | 0.0 |
| Allowed | 7.44 | 9.91 | 7.15 | 5.72 | 7.9 |
| favored | 92.56 | 90.05 | 92.85 | 94.28 | 91.94 |
| Rotamer outliers (%) | 0.51 | 0.09 | 8.99 | 0.35 | 0.18 |
| Molprobity score | 1.82 | 2.29 | 2.38 | 1.84 | 1.99 |
| EMRinger score | 3.04 | 1.61 | 2.67 | 2.28 | 2.75 |
Additional files
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Supplementary file 1
Supplementary method for the synthesis of GFB-8749.
- https://cdn.elifesciences.org/articles/60603/elife-60603-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/60603/elife-60603-transrepform-v2.docx
